Ovarian hormone and method of producing the same



rerun z ll lltl i llfil'lill tilt" il' ranlr llldred, hllount ll'aher, l t. designer to lileed it; (Carin; tron oi l lo mutte ing.

Application April Jill,

it" ti ittlilil This invention relates to the commercial production of an ovarian hormone and to the method or" ezrtractins and puriiying the same and to the resulting product and is a continua tion in part of my copending application Serial lilo. ll'5,933 tiled lviarch 16, 1927.

Various attempts have been made in the past to extract the active principle or hormone of the ovaries and to purify such extract or hormone 1 lay the elimination of all toxic and inert material so as to produce an active therapeutic product suitable for use in treating cases oi dysiuuction in the some human gland, either by injection or oral administration.

The prior attempts alone this line have, lldtlf ever, either railed to produce an active product suillcieritly purified, for human use, or Where a highly purified product has been produced. its activity has been of an uncertain nature and the process used has teen ill edapted for quantity production or has been too expensive to permit commercialized production of the hormone to sell at a price permitting general use thereof.

it is an object oi the present invention to pro duce an ovarian hormone having all the desired activity of the natural gland and. in as high or higher state oi purity than that heretofore lrnovvn, hy a process adapted for quantity production oi the product on a corrunercial seals W and at a comparatively lov. cost.

Another object of the invention is to provide the hormone in an active state as a product oi? manufacture for use either by injection or oral administration.

Another object of the invention is to provide as a product of manufacture for therapeutic uses a combination or an ovarian hormonewith the extract or hormone of other related glands with or without drugs to produces balanced ther- W apeutic compound.-

lit will he understood that the method which i. propose to use may be varied in many details to commercially produce the hormone and that the hormone may he variously combined with other medicinal products than those specifically lid or the scope of the appended claims.

In general not process comprises extracting the major portion oi the hormone from the minced. glands by the use of solvents which, while removing substantially all the hormone, Will dissolve as little as possible of the phosphatids, rats, cholesteral and other inert or toxic materials from the glands, and the subsequent. removal of the hormone from this original. extract contain-- ing also some phosphatids, fats, cholesterol, etc, Without first separately separating" these unde sired materials from the extract;

i prefer to prepare my extract from iresh whole ovaries because of the greater prohahllity of securing the entire active principles of the gland therefrom. l-lovvever, an active material having some, if not all, of the physiologic mach iestations of my product may he produced from corpus luteum, liquor iolliouli or placenta, and oi course cerresponding active honnones may he prepared from other glands.

in preparing my product, i first grind the fresh ovaries and the same with about three times their Weight of an organic solvent capable of err-- treating the hormone. The amount oi? solvent is preferably proportioned to the amount of Water in the fresh glands so as to produce a concentration of the solvent which will extract the greatest amount oi the physiologically active lipoid mate=- rial and the least amount 'oi the flats, protein, phospholipins, cholesterol and other physiologically inert substances. For example, when acetone is used for the original extraction it is desir able to use about four volumes because this, when mixed with the normal water constituent of the glands, produces approximately a ninety per cent. acetone which I have found to he the most efilcient acetone concentration for my purpose. When propyl alcohol is used, a final concentration of about seventy (to) percent. dissolves out the largest amount of the active substance with the least amount of inert material and I therefore prefer to start the extraction with about two volumes oi" propyi alcohol. With ethyl and methyl alcohol, a somewhat higher final concentration is desired for the first extraction and l have found that about three volumes of ninety-five per cent. alcohol are required, giving a final concentration after mixing With the Water of the glands of about seventy-five ((5) per cent. It will be understood that the amount of water in the fresh gland is not constant, and that the final concentration of the solvent may therefore vary slightly from the exact figures given, and that these figures are to indicate approximately the tilt l best worming conditions rather than to give Bra-- tlicrnatically exact concentrations.

1 have found that the lipoid material of the contain most, if not all, of the physiologicaliy active material and it is my purpose in this extract on step to use a solvent which will dissolve out the greatest portion of the llpoid mater'al the smallest portion of fats, cholesterol, phospholipins and inert or toxic material.

The ground whole ovaries are preferably macercted from twelve to forty-eight hours with the required amount of the solvent selected, the liquid filtered oil, the residue expressed and re-ex= e (the glands having been freed from. the water therein) using the exact concentration which recovers most of the hormone with the undesired material, as, for example, ninety (86) cent. acetone, seventy (70) per cent. propyl alcohol or about seventy-five per cent etlyl or methyl alcohol etc. The second extrac tion continued until the residue is fully extracted. The two extracts are combined, and the solvent distilled off at a low temperature and under reduced pressure and preferably recovered. traces of the solvent are removed, leaving-the lipoid material containing the hormone, together with some fats, cholesterol, phospholipins, etc., ermilsified In an aqueous solution.

My next step is designed to extract from this aqueous solution the active principle or hormone, leaving therein substantialy all of the cholesterol, phospholipins and inert or toxic matter and this may be acornplished by agitating the emulsion with an ester or other solvent for the hormone, such as chloroform, aniyl or butyl alcohol, amyl, butyl or ethyl acetate, etc, which has the property of dissolving out the hormone and rejecting the inactive materials.

The mixture from the agitation of the emulsifled aqueous solution of the lipold material with one of the solvents named above, when the agi em tion has ceased, separates into two or three layers, dependent upon the solvent used. When three layers are formed, the upper or solvent layer contains the active lipoicl with possible traces of cholesterol and phospholipins, and is free from protein, the middle layer contains most of the phospholipins and cholesterol present in the origincl extract, together with other organic material, and a portion of the solvent and water. The lower aoueous layer contains blood pigments, sal s, etc. The one or two lower layers are pref erebly dram on and the agitation with the hormono solvent repeated several times and finally the two or three layers are drawn ofi separately. lln. c"

'llcroiorm is used the lower chloroform the active hormones.

' illbll'lEd upper layers may then be washed one (1) to ten (10) per cent sodium can l .tion to remove all traces of the fatty hcspholipins, washed with water to *oditun carbonate and the solvent dis- .che residue then contains the hormone state of purity. For example, in using coliol at this step of the process, the aginiixture of the amyl alcohol and the aqueolutiou containing the lipoid material sep" erates into three layers, with the upper layer cents. the active portion or hormone. The two lower layers are then drawn off, the agitation with amyl alcohol repeated and the upper layers resulting from several repetitions of this step combined, washed with a one (1) to ten (10) per cent. sodium carbonate solution and then A with bou rcraaee with water 1 lie solvent distilled oil leaving the hormone state of purity.

If desired, the aqueous solution of the hormone very slightly alkaline before e z-- tracting the hormone from it, and the step of 33 washing the sodium carbonate omitted. The hormone may be dried carefully at this point made into tablets for oral administration or can pounded with other related therapeutic sub" stances into tablets or suspended in a suitable rier for injections.

When administered orally in this form to group of ovariectomized rats 2 milligrams or is equivalent to one rat unit and will produce oestrus in at least severity-five ('25) per cent. the rats to which it is fed, and when given in tablet form. to human beings will produce bencficial results. I believe my products to be the first ovarian hormone capable of producing demonstrable therapeutic properties on oral administration.

The active ovarian hormone is light yellow color and at room temperature is of a. warm con-= sistency. It does not give the biuret test, but a positive cholesterol reaction. It is in a big fled state of purity any or the so called ova i hormones heretofore produced and is, therefore, more concentrated'rnore active and less toxic than prior products. When dissolved in oil aqueous solutions and injected into ovariectom ized or immatur rats, it produces growth of the uterus, symptoms oi oestrus and stimulates genital organs in other ways. If the injections are continued over long period or time, changes in secondary sex characteristics, which ordinarily r accompany ovariectomy, are prevented. 1. same results are produced by feeding the active lipoid substances over a considerable period.

Before using the hormone for the treatment oi human beings by injection, is preferable to purify it further by dissolving in ninetydlve per cent alcohol, filter and discmd undissolved portion consisting largely of cholesterol, recover the alcohol and dry the residue, dissolve residue in ethyl ether, filter, evaporate the ether and dissolve residue in cotton seed or other oil, aqueous solu tions, saline the for injections. The cholesterol j also be removed by washing the hormone while suspended in seventy (70) per cent alcohol with petroleum ether, which dissolves out substantially all the cholesterol without removing a material percentage of the hormone.

In lieu of a high concentration of the initial solvent, which is diluted by the water in the i'resh glands, the freshly ground glands may be dried in a vacuum to remove the Water and the extrac tion started wi .23 concentration of the solvent which is most lent the (1m glands.

The hormone is especially recommended treatment of notions of the human ovaries and female genit organs. It is effective either when administere y injection or orally.

For clinical and e oral medical use, I find it desirable to corbthe hormone with oth medicinals elated effect on the same .3 related organs of the body to produce a complete and balanced action or stimulation of these parts of the body.

Inasmuch as the dysfiu'iction of the ovaries and genital organs is often either caused by or acconrpanied by improper functioning and deficiencies of other organs and parts or" the body, I find it desirable, in order to eiiect complete restoration of normal bodily functions to combine with the ovarian hormone. other rnedicinals having a reover of oveileotoniized rats to which it is fed.

7. The method of. concentrating and purifying the hormones of the female sex glands from corpus luteum, which o nipri es extracting the hormone fr m the fresh glands with e water and lipoici solvent, driving" oil the solvent to produce an aqueous lipoicl residue and. extracting the ho ones from the residue at least once with an miscible fat solvent, washing the hormo -e while suspended in fat solvent with a. sodium mi'honote solution and wetter, removing the fat solvent, dissolving the hormone in alcohol, filtering to remove the precipitate, removing the alcohol solvent, dissolving a further fat solvent,

8. A potent Effiacb from tissues containing the female sex hormones such as corpus luteum, ssid extract of the order obtained by treat ng fresh, corpus luteum tissue with a, Water and lipoici solvent to remove the lipoids therefrom concentrating the solution to an aqueous iipoioi materiel, treating said aqueous lipoid material with a Water immiscible fat solvent, washing with sodium carbonate, removing the fat solvent, r dissolving in alcohol and removing the precipitate, removing the alcohol solvent i'edissolving in a further fat solvent, removing the fat solvent and dissolving the hormone in a solution for misc-- tion.

FRANK R. ELDRED.

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